*Target (protein/gene name): Zinc metalloproteinase (also called lethal factor endopeptidase) *NCBI Gene # or RefSeq#: 3361711 (NCBI #) *Protein ID (NP or XP #) or Wolbachia#: 3.4.24.83 *Organism (including strain): Bacillus anthracis Etiologic Risk Group (see link below): Level 4 risk group (category A priority pathogen) */Disease Information (sort of like the Intro to your Mini Research Write up):Bacillus anthracis is a bacteria that can cause infection in the lungs, the intestinal tract, in the blood by injection, or topically on the skin. Even with treatment, inhalation of anthrax has between a 50 and 80% mortality rate. Due to its virulence it has been developed into a weapon by many countries and a treatment for it is high priority since it has so much potential as a bioterrorist weapon. The toxin that anthrax secretes is made of three parts - the lethal factor, edema factor, and protective antigen. The toxin can have continued effects in a persons body even after the bacteria itself has been killed which is why a treatment for the toxin is needed as well as antibiotics. Link to TDR Targets page (if present): n/a Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.): https://www.ncbi.nlm.nih.gov/gene/3361711 Essentiality of this protein: Essential for the virulence of the organism in the toxin that it secretes Is it a monomer or multimer as biological unit? (make prediction athttp://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): monomer with 4 domains Complex of proteins?: no Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): evidence of binding in PDB entries https://www.rcsb.org/structure/4PKT Maize, K.; Kurbanov, E.; De La Mora-Rey, T.; Geders, T.; Hwang, D.-J.; Walters, M.; Johnson, R.; Amin, E.; Finzel, B., Anthrax toxin lethal factor domain 3 is highly mobile and responsive to ligand binding. Acta Crystallogr D Biol Crystallogr2014,70 (11), 2813-2822.
--- List cost and quantity of substrate reagents, supplier, and catalog #
DMSO: $40.50 (50 mL), Sigma Aldrich, D8418
Abz/DNP-substrate: $285.00 (200 nmoles), List Biological Laboratories, Inc., 530
Structure (PDB or Homology model) -- PDB # or closest PDB entry if using homology model: 4PKT
Current Inhibitors: listed on BRENDA and DB binding Expression Information (has it been expressed in bacterial cells): E. coli BL21 (DE3) Purification Method: Ni-NTA column and Hiprep 26/60 Sephacryl S-200 HR column Image of protein (PyMol with features delineated and shown separately): *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): SNALSRYEKWEKIKQHYQHWSDSLSEEGRGLLKKLQIPIEPKKDDIIHSLSQEEKELLKRIQIDSSDFLSTEEKEFLKKL
RLMHSTDHAERLKVQKNAPKTFQFINDQIKFIINSLVPR *length of your protein in Amino Acids: 519 amino acids Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 60.35126 kDa Molar Extinction coefficient of your protein at 280 nm wavelength: 58790 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it. *CDS Gene Sequence (paste as text only): ATGAATATAAAAAAAGAATTTATAAAAGTAATTAGTATGTCATGTTTAGTAACAGCAATTACTTTGAGTG GTCCCGTCTTTATCCCCCTTGTACAGGGGGCGGGCGGTCATGGTGATGTAGGTATGCACGTAAAAGAGAA AGAGAAAAATAAAGATGAGAATAAGAGAAAAGATGAAGAACGAAATAAAACACAGGAAGAGCATTTAAAG GAAATCATGAAACACATTGTAAAAATAGAAGTAAAAGGGGAGGAAGCTGTTAAAAAAGAGGCAGCAGAAA AGCTACTTGAGAAAGTACCATCTGATGTTTTAGAGATGTATAAAGCAATTGGAGGAAAGATATATATTGT GGATGGTGATATTACAAAACATATATCTTTAGAAGCATTATCTGAAGATAAGAAAAAAATAAAAGACATT TATGGGAAAGATGCTTTATTACATGAACATTATGTATATGCAAAAGAAGGATATGAACCCGTACTTGTAA TCCAATCTTCGGAAGATTATGTAGAAAATACTGAAAAGGCACTGAACGTTTATTATGAAATAGGTAAGAT ATTATCAAGGGATATTTTAAGTAAAATTAATCAACCATATCAGAAATTTTTAGATGTATTAAATACCATT AAAAATGCATCTGATTCAGATGGACAAGATCTTTTATTTACTAATCAGCTTAAGGAACATCCCACAGACT TTTCTGTAGAATTCTTGGAACAAAATAGCAATGAGGTACAAGAAGTATTTGCGAAAGCTTTTGCATATTA TATCGAGCCACAGCATCGTGATGTTTTACAGCTTTATGCACCGGAAGCTTTTAATTACATGGATAAATTT AACGAACAAGAAATAAATCTATCCTTGGAAGAACTTAAAGATCAACGGATGCTGTCAAGATATGAAAAAT GGGAAAAGATAAAACAGCACTATCAACACTGGAGCGATTCTTTATCTGAAGAAGGAAGAGGACTTTTAAA AAAGCTGCAGATTCCTATTGAGCCAAAGAAAGATGACATAATTCATTCTTTATCTCAAGAAGAAAAAGAG CTTCTAAAAAGAATACAAATTGATAGTAGTGATTTTTTATCTACTGAGGAAAAAGAGTTTTTAAAAAAGC TACAAATTGATATTCGTGATTCTTTATCTGAAGAAGAAAAAGAGCTTTTAAATAGAATACAGGTGGATAG TAGTAATCCTTTATCTGAAAAAGAAAAAGAGTTTTTAAAAAAGCTGAAACTTGATATTCAACCATATGAT ATTAATCAAAGGTTGCAAGATACAGGAGGGTTAATTGATAGTCCGTCAATTAATCTTGATGTAAGAAAGC AGTATAAAAGGGATATTCAAAATATTGATGCTTTATTACATCAATCCATTGGAAGTACCTTGTACAATAA AATTTATTTGTATGAAAATATGAATATCAATAACCTTACAGCAACCCTAGGTGCGGATTTAGTTGATTCC ACTGATAATACTAAAATTAATAGAGGTATTTTCAATGAATTCAAAAAAAATTTCAAATATAGTATTTCTA GTAACTATATGATTGTTGATATAAATGAAAGGCCTGCATTAGATAATGAGCGTTTGAAATGGAGAATCCA ATTATCACCAGATACTCGAGCAGGATATTTAGAAAATGGAAAGCTTATATTACAAAGAAACATCGGTCTG GAAATAAAGGATGTACAAATAATTAAGCAATCCGAAAAAGAATATATAAGGATTGATGCGAAAGTAGTGC CAAAGAGTAAAATAGATACAAAAATTCAAGAAGCACAGTTAAATATAAATCAGGAATGGAATAAAGCATT AGGGTTACCAAAATATACAAAGCTTATTACATTCAACGTGCATAATAGATATGCATCCAATATTGTAGAA AGTGCTTATTTAATATTGAATGAATGGAAAAATAATATTCAAAGTGATCTTATAAAAAAGGTAACAAATT ACTTAGTTGATGGTAATGGAAGATTTGTTTTTACCGATATTACTCTCCCTAATATAGCTGAACAATATAC ACATCAAGATGAGATATATGAGCAAGTTCATTCAAAAGGGTTATATGTTCCAGAATCCCGTTCTATATTA CTCCATGGACCTTCAAAAGGTGTAGAATTAAGGAATGATAGTGAGGGTTTTATACACGAATTTGGACATG CTGTGGATGATTATGCTGGATATCTATTAGATAAGAACCAATCTGATTTAGTTACAAATTCTAAAAAATT CATTGATATTTTTAAGGAAGAAGGGAGTAATTTAACTTCGTATGGGAGAACAAATGAAGCGGAATTTTTT GCAGAAGCCTTTAGGTTAATGCATTCTACGGACCATGCTGAACGTTTAAAAGTTCAAAAAAATGCTCCGA AAACTTTCCAATTTATTAACGATCAGATTAAGTTCATTATTAACTCATAA *GC% Content for gene: 30% *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized):
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences): **
*NCBI Gene # or RefSeq#: 3361711 (NCBI #)
*Protein ID (NP or XP #) or Wolbachia#: 3.4.24.83
*Organism (including strain): Bacillus anthracis
Etiologic Risk Group (see link below): Level 4 risk group (category A priority pathogen)
*/Disease Information (sort of like the Intro to your Mini Research Write up): Bacillus anthracis is a bacteria that can cause infection in the lungs, the intestinal tract, in the blood by injection, or topically on the skin. Even with treatment, inhalation of anthrax has between a 50 and 80% mortality rate. Due to its virulence it has been developed into a weapon by many countries and a treatment for it is high priority since it has so much potential as a bioterrorist weapon. The toxin that anthrax secretes is made of three parts - the lethal factor, edema factor, and protective antigen. The toxin can have continued effects in a persons body even after the bacteria itself has been killed which is why a treatment for the toxin is needed as well as antibiotics.
Link to TDR Targets page (if present): n/a
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.):
https://www.ncbi.nlm.nih.gov/gene/3361711
Essentiality of this protein: Essential for the virulence of the organism in the toxin that it secretes
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): monomer with 4 domains
Complex of proteins?: no
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): evidence of binding in PDB entries
https://www.rcsb.org/structure/4PKT
Maize, K.; Kurbanov, E.; De La Mora-Rey, T.; Geders, T.; Hwang, D.-J.; Walters, M.; Johnson, R.; Amin, E.; Finzel, B., Anthrax toxin lethal factor domain 3 is highly mobile and responsive to ligand binding. Acta Crystallogr D Biol Crystallogr 2014, 70 (11), 2813-2822.
*EC#: 3 (hydrolase, 3.4.24.83)
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/enzyme.php?ecno=3.4.24.83
-- Show screenshot of BRENDA enzyme mechanism schematic
-- -or link (or citation) to paper that contains assay information
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2805240/
-- links to assay reagents (substrates) pages.
https://www.sigmaaldrich.com/catalog/substance/dimethylsulfoxide78136768511?lang=en®ion=US&attrlist=Assay
https://www.listlabs.com/products/buy-mapkkide-peptide-substrate-o-abzdnpfor-anthrax-lethal-factor
--- List cost and quantity of substrate reagents, supplier, and catalog #
DMSO: $40.50 (50 mL), Sigma Aldrich, D8418
Abz/DNP-substrate: $285.00 (200 nmoles), List Biological Laboratories, Inc., 530
Structure (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 4PKT
Current Inhibitors: listed on BRENDA and DB binding
Expression Information (has it been expressed in bacterial cells): E. coli BL21 (DE3)
Purification Method: Ni-NTA column and Hiprep 26/60 Sephacryl S-200 HR column
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
SNALSRYEKWEKIKQHYQHWSDSLSEEGRGLLKKLQIPIEPKKDDIIHSLSQEEKELLKRIQIDSSDFLSTEEKEFLKKL
QIDIRDSLSEEEKELLNRIQVDSSNPLSEKEKEFLKKLKLDIQPYDINQRLQDTGGLIDSPSINLDVRKQYKRDIQNIDA
LLHQSIGSTLYNKIYLYENMNINNLTATLGADLVDSTDNTKINRGIFNEFKKNFKYSISSNYMIVDINERPALDNERLKW
RIQLSPDTRAGYLENGKLILQRNIGLEIKDVQIIKQSEKEYIRIDAKVVPKSKIDTKIQEAQLNINQEWNKALGLPKYTK
LITFNVHNRYASNIVESAYLILNEWKNNIQSDLIKKVTNYLVDGNGRFVFTDITLPNIAEQYTHQDEIYEQVHSKGLYVP
ESRSILLHGPSKGVELRNDSEGFIHEFGHAVDDYAGYLLDKNQSDLVTNSKKFIDIFKEEGSNLTSYGRTNEAEFFAEAF
RLMHSTDHAERLKVQKNAPKTFQFINDQIKFIINSLVPR
*length of your protein in Amino Acids: 519 amino acids
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 60.35126 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength: 58790
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
ATGAATATAAAAAAAGAATTTATAAAAGTAATTAGTATGTCATGTTTAGTAACAGCAATTACTTTGAGTG
GTCCCGTCTTTATCCCCCTTGTACAGGGGGCGGGCGGTCATGGTGATGTAGGTATGCACGTAAAAGAGAA
AGAGAAAAATAAAGATGAGAATAAGAGAAAAGATGAAGAACGAAATAAAACACAGGAAGAGCATTTAAAG
GAAATCATGAAACACATTGTAAAAATAGAAGTAAAAGGGGAGGAAGCTGTTAAAAAAGAGGCAGCAGAAA
AGCTACTTGAGAAAGTACCATCTGATGTTTTAGAGATGTATAAAGCAATTGGAGGAAAGATATATATTGT
GGATGGTGATATTACAAAACATATATCTTTAGAAGCATTATCTGAAGATAAGAAAAAAATAAAAGACATT
TATGGGAAAGATGCTTTATTACATGAACATTATGTATATGCAAAAGAAGGATATGAACCCGTACTTGTAA
TCCAATCTTCGGAAGATTATGTAGAAAATACTGAAAAGGCACTGAACGTTTATTATGAAATAGGTAAGAT
ATTATCAAGGGATATTTTAAGTAAAATTAATCAACCATATCAGAAATTTTTAGATGTATTAAATACCATT
AAAAATGCATCTGATTCAGATGGACAAGATCTTTTATTTACTAATCAGCTTAAGGAACATCCCACAGACT
TTTCTGTAGAATTCTTGGAACAAAATAGCAATGAGGTACAAGAAGTATTTGCGAAAGCTTTTGCATATTA
TATCGAGCCACAGCATCGTGATGTTTTACAGCTTTATGCACCGGAAGCTTTTAATTACATGGATAAATTT
AACGAACAAGAAATAAATCTATCCTTGGAAGAACTTAAAGATCAACGGATGCTGTCAAGATATGAAAAAT
GGGAAAAGATAAAACAGCACTATCAACACTGGAGCGATTCTTTATCTGAAGAAGGAAGAGGACTTTTAAA
AAAGCTGCAGATTCCTATTGAGCCAAAGAAAGATGACATAATTCATTCTTTATCTCAAGAAGAAAAAGAG
CTTCTAAAAAGAATACAAATTGATAGTAGTGATTTTTTATCTACTGAGGAAAAAGAGTTTTTAAAAAAGC
TACAAATTGATATTCGTGATTCTTTATCTGAAGAAGAAAAAGAGCTTTTAAATAGAATACAGGTGGATAG
TAGTAATCCTTTATCTGAAAAAGAAAAAGAGTTTTTAAAAAAGCTGAAACTTGATATTCAACCATATGAT
ATTAATCAAAGGTTGCAAGATACAGGAGGGTTAATTGATAGTCCGTCAATTAATCTTGATGTAAGAAAGC
AGTATAAAAGGGATATTCAAAATATTGATGCTTTATTACATCAATCCATTGGAAGTACCTTGTACAATAA
AATTTATTTGTATGAAAATATGAATATCAATAACCTTACAGCAACCCTAGGTGCGGATTTAGTTGATTCC
ACTGATAATACTAAAATTAATAGAGGTATTTTCAATGAATTCAAAAAAAATTTCAAATATAGTATTTCTA
GTAACTATATGATTGTTGATATAAATGAAAGGCCTGCATTAGATAATGAGCGTTTGAAATGGAGAATCCA
ATTATCACCAGATACTCGAGCAGGATATTTAGAAAATGGAAAGCTTATATTACAAAGAAACATCGGTCTG
GAAATAAAGGATGTACAAATAATTAAGCAATCCGAAAAAGAATATATAAGGATTGATGCGAAAGTAGTGC
CAAAGAGTAAAATAGATACAAAAATTCAAGAAGCACAGTTAAATATAAATCAGGAATGGAATAAAGCATT
AGGGTTACCAAAATATACAAAGCTTATTACATTCAACGTGCATAATAGATATGCATCCAATATTGTAGAA
AGTGCTTATTTAATATTGAATGAATGGAAAAATAATATTCAAAGTGATCTTATAAAAAAGGTAACAAATT
ACTTAGTTGATGGTAATGGAAGATTTGTTTTTACCGATATTACTCTCCCTAATATAGCTGAACAATATAC
ACATCAAGATGAGATATATGAGCAAGTTCATTCAAAAGGGTTATATGTTCCAGAATCCCGTTCTATATTA
CTCCATGGACCTTCAAAAGGTGTAGAATTAAGGAATGATAGTGAGGGTTTTATACACGAATTTGGACATG
CTGTGGATGATTATGCTGGATATCTATTAGATAAGAACCAATCTGATTTAGTTACAAATTCTAAAAAATT
CATTGATATTTTTAAGGAAGAAGGGAGTAATTTAACTTCGTATGGGAGAACAAATGAAGCGGAATTTTTT
GCAGAAGCCTTTAGGTTAATGCATTCTACGGACCATGCTGAACGTTTAAAAGTTCAAAAAAATGCTCCGA
AAACTTTCCAATTTATTAACGATCAGATTAAGTTCATTATTAACTCATAA
*GC% Content for gene: 30%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**